Author: Alexis Prybutok
Advisor: Jennifer Maynard, Ph.D.
Department: McKetta Department of Chemical Engineering
Institution: The University of Texas at Austin
Production of antibody fragments for testing synergy between anti-Pertussis toxin antibodies
In recent years, the incidence of pertussis, or whooping cough, increased worldwide, particularly in infants.1 This may be in part due to the introduction of a new acellular vaccine, which is less effective long term, enabling pathogen growth in adults and transfer to infants.2 Although generally mild in adults, pertussis can cause hospitalization and/or death in infants too young to be vaccinated. Our lab is developing a therapeutic consisting of two anti-pertussis toxin antibodies that help fight the disease by injection into sick individuals.
Pertussis toxin (PTx), secreted by Bordetella pertussis, causes the majority of systemic symptoms that result in severe disease.3.4 PTx consists of an A (“active”) subunit that modifies proteins inside of cells and a B (“binding”) subunit that allows the toxin to enter host cells. Two previously identified antibodies exhibit a high affinity for PTx: 1B7 and 11E6. The 1B7 antibody binds the A subunit, neutralizing the catalytic activity while 11E6 binds two sites on the B subunit, inhibiting toxin binding onto extracellular receptors.2,3 We have previously demonstrated that a binary mixture of 1B7 and 11E6 neutralizes the toxin most effectively, presumably due to their complementary mechanisms.
We are interested in further investigating the synergy of 1B7 and 11E6. To do this, we propose using antibody fragments (fabs) consisting of only one antibody “arm”, which is a monovalent format that simplifies the antibody:toxin binding interaction. This allows testing synergistic ratios of 1B7 and 11E6 that could not be tested using the full length antibodies. The full-length1B7 plasmid was truncated and transfected into CHO cells. 1B7 fab secreted by the CHO cells was collected and purified by affinity chromatography. After production, antibody identity was confirmed by SDS-PAGE. PTx binding of the 1B7 fab was then compared to that of the full length 1B7 antibody using an ELISA. Currently, the fab is being tested in vitrovia CHO cell assays to further asses the synergy of 1B7 and 11E6.
1. Cherry, J.; Brunell, P.; Golden, G.; Karzon, D.; Report of the Task Force on Pertussis and Pertussis Immmunization—1988. Pediatrics. 1988, 81, 933-984.
2. Sutherland, J.; Maynard, J.; Characterization of a Key Neutralizing Epitope on Pertussis Toxin Recognized by Monoclonal Antibody 1B7. Biochemistry, 2009, 48, 11982-11993.
3. Sato, H.; Sato, Y.; Protective activities in mice of monoclonal antibodies against pertussis toxin. Infect.Immun.. October 1990, 58, 3369-3374.
4. Sato, H.; Sato, Y.; Ito, A.; Ohishi, I.; Effect of monoclonal antibody to pertussis toxin on toxin activity.Infect. Immun. April 1987, 55, 909-915.
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